JP2000514795A - Complex of thrombin inhibitor and endogen carrier - Google Patents

Abstract

Novel compounds comprising chemically reactive intermediates which can react with available reactive functionalities on blood components to form covalent linkages, where the resulting covalently-bound conjugates are found to have thrombin inhibition activity are provided. Specifically, the thrombin inhibitor compounds of the present invention are derivatives of the known thrombin inhibitor argatroban, which can be covalently linked to chemically reactive functionalities on various blood components. The conjugated thrombin inhibitors thereby have extended lifetimes in the bloodstream, as compared to the unconjugated parent drug, and are, therefore, capable of maintaining thrombin inhibitory activity for extended periods of time as compared to the unconjugated parent drug. Also provided herein are methods for inhibiting thrombin activity in vivo comprising administering to the bloodstream of a mammalian host the novel compounds of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION          Complex of thrombin inhibitor and endogen carrierTechnical field   The field of the invention is the extended lifespan of a bioactive agent in a mammalian host, more particularly The prolonged lifespan of an inhibitor of thrombin activity in a mammalian host.background   Many attempts have been made to obtain new and improved drugs for treating thrombosis. Had been done. N^Two-(P-tolylsulfonyl) -L-arginine ester is used. Are one type of drug that can be used, and these can be effective in lysing blood clots. Known (see U.S. Pat. No. 3,622,615 issued Nov. 23, 1971 When). Characterized as a highly specific inhibitor of thrombin for thrombosis control Another family of compounds found to be beneficial to^Two-Dancil-L-A Luginine esters or amides (US Pat. No. 3,978,045) and many N^Two-Ant Sulphonyl-L-argininamide. In addition, for the treatment of thrombosis in mammals Another compound that has been found to be particularly useful is 1- [5- [aminoiminomethyl) Amino] -1-oxo-[[(1,2,3,4-tetrahydro-3-methyl-8 -Quinolinyl) sulfonyl] -amino] pentyl] -4-methyl-2-piperidi Carboxylic acid, a compound commonly known as argatroban.   The above compounds are associated with abnormal thrombosis by suppressing thrombin activity in vivo Although they have been shown to be beneficial in the treatment of diseases, their therapeutic utility is Many of the products are rapidly degraded and / or cleared from the host vasculature after administration Limited by the fact that. As such, extended time The thrombin inhibitors described above for continuous thrombin inhibitory activity over a period of time Can be administered as a large bolus administered at periodic time intervals or as a depot containing the drug. It must be administered by providing the agent. However, unfortunately, Administration of large boluses with large periodic intervals often over extended periods of time Subtherapeutic dosage of the drug, and then significantly above the desired therapeutic level Resulting in an acceptable dosage. The latter can often be accompanied by serious side effects. Furthermore, various Pumps and biodegradable and non-biodegradable capsules for extended periods of time These devices have been devised for drug delivery over time. These profiles can have various drawbacks, for example, And / or interfere with their release of the active agent.   Therefore, there is an interest in providing improved therapies associated with thrombin suppression. You.   Summary of the Invention   Can react with available reactive functional groups on blood components to form covalent bonds New compounds comprising chemically reactive intermediates are provided, in which case the resulting covalent bonds It can be seen that the complex has thrombin inhibitory activity. Specifically, the compounds of the present invention Is a known thrombin inhibitor, a compound also known as argatroban -1- [5- (aminoiminomethyl) amino] -1-oxo-2-[[(1,2 , 3,4-Tetrahydro-3-methyl-8-quinolinyl) sulfonyl] -amino ] Pentyl] -4-methyl-2-piperidinecarboxylic acid You. The derivatized argatroban molecules of the present invention have an active thrombin inhibitor moiety. Includes covalently bonded to reactive functional groups of various blood components by chemically reactive groups become. Thereby, the complex thrombin inhibitor molecule is compared with the unbound parent drug. Have an extended lifespan in the bloodstream and therefore an extended time compared to the unbound parent drug Thrombin inhibitory activity can be maintained over a period of time.   Also, the above derivatized thrombin in combination with a pharmaceutically acceptable carrier. Administering a Novel Compound of the Invention to a Composition Containing an Inhibitor Molecule and the Blood Stream of a Mammalian Host And a method for suppressing thrombin activity in vivo.   BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the processes involved in the synthesis of various derivatized thrombin inhibitor molecules of the present invention. It is a schematic diagram of a process.   FIG. 2 shows that human serum albumin (HSA) alone (“●”) was pre-reacted (prequenc- hed) HSA treated with hydroxylamine-treated argatroban-C6 NHS ester ( “△”), HSA treated with argatroban-C12 NHS ester which had been pre-reacted “*”), HSA (“◆”) or algatroban-C6 NHS ester Sandwich ELIS using HSA ("X") reacted with gatroban-C12NHS ester 4 is a graph showing the results obtained in the A assay. The result is the absorbance at 490 mn (“O D 490 ”) vs. HSA concentration (nanogram / milliliter,“ ng / ml ”) .   Figure 3 shows rabbit serum albumin (RSA) alone ("△"), pre-reacted argato RSA (“■”) or Argatroban-C6 NHS reacted with Roban-C6 NHS ester Obtained in a sandwich ELISA assay using RSA (“◆”) reacted with an ester 9 is a graph showing the results. Results are absorbance at 490nm ("OD 490") vs RSA Expressed as concentration (nanogram / milliliter, "ng / ml").   Figure 4 shows human glycophorin A (hGPA) alone ("△"), pre-reacted argato HGPA (“■”) or argatroban-C6 N treated with Roban-C6 NHS ester In a sandwich ELISA assay using hGPA (“◆”) treated with HS ester It is a graph which shows the obtained result. Results are absorbance at 490 nm ("OD 490") v s Expressed as hGPA concentration (nanogram / milliliter, “ng / ml”).   Fig. 5 shows HSA alone ("■") and argatroban-C6 NHS ester that had been pre-reacted. HSA treated with “*”, HSA treated with argatroban-C6 NHS ester (1: 5 HSA reacted with argatroban-C6 NHS ester (1: 1 dilution) (“◆”) 000 dilution) (a group showing the results obtained in a sandwich ELISA assay using “X”). It is rough. The result is absorbance at 490 nm (“OD 490”) vs HSA concentration (nanogra Per milliliter, expressed as "ng / ml").   FIG. 6 shows that hGPA alone (“△”) was stopped using the 5F4 monoclonal antibody, and the pre-reaction was stopped. (“■”) or Argato treated with argatroban-C6 NHS ester Sandwich ELISA using hGPA ("◆") reacted with Roban-C6 NHS ester 4 is a graph showing the results obtained in the assay. The result is the absorbance at 490 nm (“OD  490 ”) expressed as hGPA concentration (nanogram / milliliter,“ ng / ml ”) .   FIG. 7 shows that hGPA alone (“△”) using 3C10 monoclonal antibody, (“■”) or Argato treated with argatroban-C6 NHS ester Sandwich ELISA using hGPA ("◆") reacted with Roban-C6 NHS ester 4 is a graph showing the results obtained in the assay. The result is the absorbance at 490 nm (“OD  490 ”) expressed as hGPA concentration (nanogram / milliliter,“ ng / ml ”) .   FIG. 8 shows that hGPA alone (“△”) using 3E4 monoclonal antibody, pre-reaction was stopped. (“■”) or Argato treated with argatroban-C6 NHS ester Sandwich ELISA using hGPA ("◆") reacted with Roban-C6 NHS ester 4 is a graph showing the results obtained in the assay. The result is the absorbance at 490 nm (“OD  490 “) vs hGPA concentration (nanogram / milliliter,“ ng / ml ”) .   FIG. 9 shows RSA alone (“O”) or reacted with argatroban-C6 NHS ester Shows the results obtained in a thrombin inhibitor assay using RSA ("●") It is a graph. Results are expressed as% inhibition of thrombin vs. amount of RSA used (nM). It is.   Figure 10 shows that thrombin alone ("O") or argatroban-C6 NHS ester Thrombin inhibitor assay using erythrocyte ghosts ("+") It is a graph which shows the obtained result. Position of the graph where the compound erythrocyte ghost was used Positions are indicated by the respective symbols ("+"), but these positions are 200 μg, 450 μg and And three parallel lines representing the addition of 600 μg ghost / ml (top to bottom of graph). At). Results are expressed as thrombin activity vs. number of minutes at 21 ° C .   FIG. 11 shows a novel thrombin inhibitor of the present invention that delays thrombin-induced platelet aggregation. A graph showing the results obtained demonstrating the ability of bitter (when covalently attached to RSA). It is. Data are RSA alone (“RSA”) or argatroban-C6 NHS ester Concentration of RSA (“RSA-Ar”) covalently bound by molecule (μg / ml) vs. 50% platelet aggregation Expressed in minutes. Assays were performed in duplicate and each assay was performed in duplicate. The assay is shown as a separate column at each concentration tested.Detailed description of the invention   Disorders associated with abnormal thrombosis due to suppression of mammalian proteins and thrombin Methods and compositions are provided for treating patients having. The method is known thrombin in New thrombin inhibitor compounds, including derivatives of the inhibitor argatroban, are used. The new thrombin inhibitor compounds are available for use in various blood components. Reacts with reactive functional groups, thereby derivatizing the derivatized thrombin inhibitor molecule. Has a chemically reactive group that is covalently bonded to blood components. The chemically reactive group is thrombin When the inhibitor moiety is bound to a blood component, the thrombin inhibitor moiety At a site that retains a substantial portion of the inhibitory activity of the parent compound. Therefore, longevity The covalent attachment of the subject thrombin inhibitor molecule to the blood component of life results in the host's blood The useful life of the thrombin inhibitor in the tubing is greatly increased.   Various sites that can react with chemically reactive groups of the subject thrombin inhibitor molecule Containing cells, especially erythrocytes and platelets, and proteins such as IgG and IgM. Immunoglobulins, serum albumin, ferritin, steroid binding proteins, Transferrin, thyroxine binding protein, α-2-macroglobulin, etc. No. These tampers with which derivatized thrombin inhibitor compounds react Proteins (these are not long-lived) are generally cleared from the host within about 3 days As a result, the above proteins (including cell proteins) were measured from about 1-3 hours after administration. At least 3 days, especially for the half-life, based on the determined blood concentration. Always survives at least 4 days, 5 days or more, usually not more than 60 days, usually more than 30 days May remain.   In most cases, reactions are based on mobile components in the blood, especially blood proteins and cells, Blood proteins and red blood cells. "Move" means that the component is generally longer than 5 minutes Of course, it has a fixed position for an extended period of time, usually not more than one minute. But some of the blood components are relatively stationary over an extended period of time Means that you may. Initially, a relatively heterogeneous collection of functionalized proteins and cells There will be a group. However, in most cases, the population within a few days May vary substantially from the initial population, depending on the half-life of the functionalized protein in Would. Therefore, usually within about 3 days or longer, IgG is the predominant functionalizer in the bloodstream. Will be protein.   Usually, by day 5 after administration, IgG, serum albumin and red blood cells It is at least about 60 mol%, usually at least about 75 mol% of the components, and contains IgG, IgM (actual). ) And serum albumin has at least about 50 of the non-cellular complex components. %, Usually at least about 75 mol%, more usually at least about 80 mol%.   The derivatized thrombin inhibitor molecule of the present invention often comprises Will have an expression.   Where:   Y is 2 to 30, more usually 2 to 18, preferably 6 to 12 atoms in the chain, especially carbon , Oxygen, phosphorus and nitrogen, and more particularly carbon and oxygen-containing linking groups. Y is alkylene, oxyalkylene, or polyoxyalkylene, preferably Alkyl having 2-15, more preferably 6-12, carbon atoms in the alkyl chain. Chain, etc., and   Z is a chemically reactive group or a precursor of a chemically reactive group, for example, carboxy, Xyesters (where the ester group has 1-8, more usually 1-6 carbon atoms Atomic groups), especially physiologically acceptable leaving groups (which are associated with amino groups in aqueous systems). Activating carboxycarbonyl for the reaction), for example, N-hydroxys Succinimide, isocyanate, thiol ester, thionocarboxylic acid ester , Imino esters, mixed acid anhydrides such as carbodiimidic anhydride, And phosphoryl esters.   The activatable precursor is usually a non-oxo-carbonyl group (its sulfur analog and nitrogen). Analogs), for example, thiono and thiolic acids and esters and iminoes A functional group that can be directly modified to provide a ter or active functional group, e.g., cyano There will be.   For covalent attachment of the derivatized thrombin inhibitors of the present invention to chemically reactive groups Reactive functional groups available in proteins are mainly amino groups, carboxyl groups and And a thiol group. All of these are the chemical reactivities of thrombin inhibitors Can be used as a target for the group, but in most cases, the linkage to the amino group Will be used with the formation of bonds. To form an amide bond, Various active carboxyl groups, especially esters, may be used as the reactive group. In some cases, the hydroxyl moiety is physiologically tolerated at the required level. Some differences The most convenient is the use of N-hydroxyl Succinimide (NHS) and N-hydroxysulfosuccinimide (sulfo-NHS ) But other alcohols (which are functional in aqueous media such as blood) May also be used. In some cases, special reagents such as azides, di Azo, carbodiimide anhydride, hydrazine, dialdehyde, thiol group, or Amines, esters, imines, thioethers, disulfides to form amides And substituted amines can be used. Usually, the covalent bond formed is Should be able to maintain during the life of the blood component, unless intended to be a site .   Optionally, the subject conjugates can also be used to convert blood to a derivatized thrombin inhibitor of the invention. Together with the derivatized thrombin inhibitor to the reactive functional group of the blood component. Prepared in vitro by allowing covalent binding and then returning the complex blood to the host obtain. In addition, the above preparation may also initially involve individual blood components or a limited number of components, For example, purifying red blood cells, immunoglobulins, serum albumin, etc., By combining the components with a chemically reactive thrombin inhibitor in vitro. obtain. The functionalized blood or blood components are then returned to the host for therapeutic treatment of the subject The complex can be given in vivo. Also, blood is processed and clots during in vitro handling. Can be prevented.   Derivatized thrombin inhibitor versus blood if the conjugate is prepared in vitro The ratio of the components is based on whether whole blood or just purified components are derivatized thrombin-in. It will vary widely depending on whether it is used as a binding site for inhibitors. all Usually about 38 micrograms / ml to about 300 micrograms each to react with blood Per ml of derivatized thrombin inhibitor to blood, while Rombin Inhibitor Vs. 10⁹The cell ratio is about 7.6 micrograms to about 300 micrograms Gram, while derivatized thrombin inhibitor vs. 1 mg protein The quality ratio will be around 38 micrograms to about 300 micrograms.   The properties of thrombin inhibitor compounds can be either random binding to long-lived blood components or It may provide varying degrees of targeted binding of blood components to a restricted class. Random In some cases, the distribution of the thrombin inhibitor compound is an activity for binding blood components. Relative proportions of sex sites, modes of administration and what thrombin inhibitors may have A priority meeting. For target binding, thrombin inhibitor compounds Part preferentially non-covalently binds to one or more sites present in one or more blood components Group. For this purpose, it is known to complex with special blood components. Objects that have been used may be used. It also gives the desired blood component association spectrum Establish combinatorial libraries and screens for library members It may be made. In most cases, a random combination will be used.   To the extent target binding is used, the choice of long-lived blood components depends on the desired longevity of the drug. Availability of blood components for binding to life and derivatized thrombin inhibitors Will be affected, at least in part.   Long-lived blood components should be at least about 12 hours, usually at least about 48 hours, and preferably At least about 5 days, preferably at least about 10 days, more preferably at least about 20 days Has a half life of more than days. Generally, the half-life is determined by the radioisotope (eg,¹³¹I,^{12 Five} I, Tc, 5¹Cr^ThreeH) or fluorescent dye and compound intravenously Measured by continuous measurement of whole blood, plasma or serum levels of the compound after injection Is determined. Red blood cells (half life about 60 days), platelets (half life about 4-7 days), vascular system Lining endothelial cells, and long-lived serum proteins such as albumin, Lloyd binding protein, ferritin, α-2-macroglobulin, transfer Phosphorus, thyroxine binding proteins, immunoglobulins, especially IgG and the like. In addition to a favorable half-life, the subject component allows for the binding of a therapeutically effective amount of a compound of the present invention. Preferably, the cell count or concentration is sufficient to achieve Cell longevity For liquid components, a cell count of at least 2,000 / μl and at least 1 μg / m l, usually at least about 0.01 mg / ml, more usually at least about 1 mg / ml of serum protein Quality concentration is preferred.   The long-lived blood component to which the subject derivatized thrombin inhibitor binds is vascular There are many in the system. Platelets are about 1-4x10^Five/ μl, while red blood cells are about 4-6x Ten⁶/ μl. Cells have a long half-life and bind to cell surface membrane proteins. It is clear that the case does not result in endocytosis. Preferred cells are hair Widely distributed in ducts and tissues and identified on the cell surface associated with their specific differentiation Expression of the binding site. In vivo administration of a subject derivatized thrombin inhibitor In addition, in the case of red blood cells and platelets, these cells are easily recovered and It may be combined with the complex in an itro and then administered to the host. Cells are usually autologous Or allogeneic, but in some cases even more heterogeneous.   Preferred erythrocyte binding site-containing molecules include glycophorins A, B and C, and Zas blood group antigens. Preferred erythrocyte binding sites are at least 1,000, preferably at least 10,000, more preferably at least 100,000 copies Abundantly expressed in red blood cells, preferably at least about 0.5 nm above the bilayer surface Derivatized thrombin inhibitor, preferably tethered at least 1 nm -Does not promote cell transformation by itself when the molecule is bound to cells (eg, The linkage will be chosen not to be a major component of the cytoskeleton). Erythrocyte surface sugar The binding site for protein glycophorin A and the erythrocyte binding site containing sialic acid are preferred. This is an example of a binding site. Preferred platelet binding sites are GPIIa, GPIIb, GPIIIa And GPIV. Desirably, after binding to the target, a long-lived blood component, e.g., No red blood cell or platelet deformation occurs.   The derivatized thrombin inhibitor of the present invention is usually administered as a bolus, It may be introduced gradually over time, such as by injection using a metering flow meter. Also so much Although not preferred, blood is removed from the host, processed ex vivo, and returned to the host. You may. The derivatized thrombin inhibitor is a physiologically acceptable medium, for example, Deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other Alcohol, plasma, protein solution, mannitol, aqueous glucose, alcohol Administration, in vegetable oils, vegetable oils and the like. As other additives that may be included A buffer (where the medium is generally buffered at a pH in the range of about 5-10, Buffering agents generally range in concentration from about 50-250 mM), salt (where the salt concentration is Generally in the range of about 5 to 500 mM), and physiologically acceptable stabilizers and the like. The composition may be lyophilized for convenient storage and transport.   The subject derivatized thrombin inhibitors are most often administered parenterally; For example, it may be administered intravascularly (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), etc. U. Administration may be by blood transfusion where appropriate. In some cases, active If the reaction of the functional groups is relatively slow, administration may be oral, nasal, rectal, transdermal or aerosol Where the nature of the complex allows metastasis to the vasculature. Normal A single injection is used, but more than one injection may be used if desired. Derivatized thrombin inhibitors include syringes, trocars, catheters, etc. Administration may be by any convenient means. The special mode of administration is administered Dosage, single bolus or continuous administration. Would. Preferably, the administration is intravascular, in which case the site of introduction is Insignificant, but not rapid, sites of blood flow, for example, intravenous, peritoneal or central vein It is preferred that Dosing is paired with a sustained release technique or a protective matrix. Other routes may be used. The purpose is that the subject compound reacts with blood components Is to be effectively distributed in the blood. Complex concentration is wide And will generally range from about 1 pg / ml to 50 mg / ml. Intravascular administration Weighing is generally from about 0.1 mg / ml to about 10 mg / ml, usually from about 1 mg / ml to about 5 mg / ml. Would be a range.   The manner in which the derivatized thrombin inhibitors of the present invention are made It will vary widely depending on the nature of the various elements involved. The synthesis method is simple, It will be chosen to give high yields and to enable highly purified products U. Usually, a chemically reactive group is created in the last step, for example, a carboxyl group Esterification to produce the active ester would be the last step in the synthesis. Departure Light An exemplary method for producing a derivatized thrombin inhibitor of is shown in FIG.   Long-lived components of blood, for example, immunoglobulins, serum albumin, erythrocytes and small blood Several advantages result from coupling to the plate. Thrombin tampa Quality control is extended over days to weeks. Only dosing during this time Need to be applied to Active compound is mainly bound to large molecules Can achieve even greater specificity, in which case it is probably absorbed into the cell and its It does not interfere with other physiological processes.   The blood of a mammalian host can be monitored more than once for the presence of a thrombin inhibitor. You. By taking a part or sample of the host's blood, Bitter is now bound to long-lived blood components in amounts sufficient to be therapeutically active Or not, followed by measuring the level of thrombin inhibitory activity in the blood . Also, if desired, thrombin inhibitor molecules bind to any of the blood components Can be measured.   The derivatized thrombin inhibitors of the present invention can be used for a variety of different applications. There will be. For example, the novel thrombin inhibitors of the present invention are associated with abnormal thrombosis. In the treatment and / or prevention of certain diseases. Subject thrombin inhibitor molecule Reduces thrombus formation, accelerates dissolution of existing thrombi, maintains blood circulation, and / or Or beneficial to improve. Also, the subject derivative molecule may be, for example, Used in the treatment of muscle infarction, leg arteritis, deep vein thrombosis, pulmonary embolism, etc. There will be.   The following examples are provided by way of illustration, and not by way of limitation.   Experiment Example 1 :Synthesis of derivatized thrombin inhibitor molecules with extended lifetime   Materials and Methods: Various Derivatized Extended Life Thrombin Inhibitors The synthesis of the-(argatroban derivatized) molecule is illustrated in FIG. 1 and described below. .   (1)Synthesis of methyl 12-aminododecanoate (compound YS-004-17)   Suspension of 12-aminododecanoic acid (5.00 g, 23.3 mmol) in anhydrous MeOH (90 mL) Hydrogen chloride gas was introduced for 25 minutes, during which time the suspension became clear. Was. The solution was then stirred at room temperature for 3 hours and the solvent was removed in vacuo. White solid Residue H_TwoDissolved in O. Solid NaHCO_ThreeWas added to the solution to neutralize to pH 8-9. like this The resulting white precipitate was collected by vacuum filtration (5.20 g) (yield: 98%).   (2)Synthesis of methyl 6-methylhexanoate (compound YS-004-58)   To a suspension of 6-aminocaproic acid (3.00 g, 22.9 mmol) in anhydrous MeOH (60 mL) , Hydrogen chloride gas was introduced over 25 minutes, during which time the suspension became clear . The solution was then stirred at room temperature for 5 hours and the MeOH was removed in vacuo. Residue in THF Recrystallization gave a white solid (3.10 g) (yield: 75%).   3.Synthesis of argatroban-C12 methyl ester (compound YS-004-67)   Argatroban monohydrate (300 mg, 0.570 mmol) and methyl acetate in anhydrous DMF (15 mL) Cyl 12-aminododecanoate (Compound YS-004-17) (132 mg, 0.576 mmol) To the solution was added HBTU (258 mg, 0.680 mmol). Stirring continued at room temperature for 16 hours . The precipitate formed during the reaction was filtered off and DMF was removed by vacuum distillation. Solvent CH_Two Cl_Two/ MeOH / NH_ThreeThe residue was purified by preparative TLC and titled using (90/10/1, v / v) The compound was obtained (140 mg) (yield: 28%).¹HNMR (MeOH-d4, 300MHz)   Four.Synthesis of argatroban-C6 methyl ester (compound YS-015-13)   Argatroban monohydrate (100 mg, 0.190 mmol) and methyl in anhydrous DMF (5 mL) 6-aminohexanoate hydrochloride (compound YS-004-58) (35 mg, 0.193 mmol) To the solution of was added triethylamine (23 mg, 0.23 mmol) followed by HBTU (86 mg, 0.23 mmol) was added. Stirring was continued at room temperature for 20 hours. The reaction The precipitate formed therein was filtered off and DMF was removed by vacuum distillation. Solvent CH_TwoCl_Two/ MeOH / N H_ThreeThe residue was purified by preparative TLC using (90/10/1, v / v) to give the title compound. 78 mg) (yield: 53%).¹HNMR (MeOH-d4,300MHz)   Five.Synthesis of argatroban-C12 free acid (compound YL-015-10)   Compound YS-004-67 (225 mg, 0.2 mL) in MeOH (4.2 mL) and 1 M aqueous NaOH (0.87 mL) (60 mmol) was stirred at room temperature for 24 hours. Cloudy solution with MeOH removed in vacuo And add H to this until the solution is clear_TwoO was further added. PH the solution with 1N HCl Acidify to 3.0 while cooling to 0 ° C. to give a white precipitate, which is The title compound was obtained (105 mg). The filtrate was extracted with n-butanol (4 x 4 mL). Was. Combine butanol solution with H_TwoWash with O (to pH 5.0) and vacuum to H_TwoAzeotropic with O And concentrated to give another portion of the compound (58 mg) (85% yield).¹HNMR (MeOH-d4,300 MHz)   6.Synthesis of argatroban-C6 free acid (compound YL-015-15)   Compound YL-015-13 (124 mg, 0.159 in MeOH (3 mL) and 1 M aqueous NaOH (0.55 mL) Mmol) was stirred at room temperature for 33 hours. The reaction was performed to prepare compound YL-015-10. (76 mg) (yield: 73%). ).¹HNMR (MeOH-d4, 300MHz)   7.Synthesis of argatroban-C12 NHS ester (compound YL-015-11)   Compound YL-015-10 (40 mg, 0.054 mmol) and N-hydroxysuccinimide (13 mg, 0.11 mmol) in diisopropylethylamine (7.4 mg, 0.057 Rimole) was added, followed by HBTU (43 mg, 0.11 mmol). The reaction mixture The mixture was stirred at room temperature for 36 hours. DMF is removed by vacuum distillation and the residue is MeOH (4 mL) Was dissolved. The MeOH solution was filtered to remove insolubles, the filtrate was concentrated in vacuo and the residue was Dissolved in a minimum amount of MeOH. Then H_TwoO to induce precipitation, The precipitate was dried in vacuo to give the title compound (19 mg) (yield: 42%).   Use EDC as the coupling reagent as the reaction yield is exemplified below After that, it was improved. Compound YL-015-10 (40 mg, 0 mg) in anhydrous DMF (3 mL) .054 mmol) and N-hydroxysuccinimide (13 mg, 0.11 mmol) To was added EDC (31 mg, 0.162 mmol). The solution was stirred at room temperature for 24 hours . DMF was removed by vacuum distillation and the residue was further dried under high vacuum. Then the residue Dissolve in a minimum amount of MeOH (0.12 mL) and add H_TwoO (3.2 mL) was added to induce precipitation. Precipitation H_TwoWashed with O (3 x 0.8 mL) and dried in vacuo to give a solid product (31 mg) (Yield: 69) %).¹HNMR (MeOH-d4,300MHz)   8.Synthesis of argatroban-C6 NHS ester (compound YL-015-35)   Compound YL-015-15 (78 mg, 0.12 mmol) and N-hydroxy in anhydrous DMF (6 mL) EDC (72 mg, 0.38 mmol) in a solution of succinimide (29 mg, 0.25 mmol) Was added. The solution was stirred at room temperature for 20 hours and DMF was removed by vacuum distillation. Dissolve the residue in a minimum amount of MeOH (0.4 mL) and add H_TwoO (1.2 mL) was added to induce precipitation . H precipitate_TwoWashed with O (3 x 0.7 mL) and dried in vacuo to give a solid product. Solid raw The product was further purified by recrystallization from acetone / ether (1/1, v / v) to give a white solid. The product was obtained (62 mg) (yield: 69%).¹HNMR (MeOH-d4, 300MHz)   9.Synthesis of argatroban-C12 sulfo-NHS ester (compound YL-015-23)   Compound YL-015-10 (10.0 mg, 13.5 μmol), sulfo-NHS (6.2 mg, 28.3 μmol) And EDC (8.2 mg, 42.5 μmol) were dissolved in anhydrous DMF (0.8 mL). The reaction mixture After stirring at room temperature for 2 days, DMF was removed by vacuum distillation. Remove the residue with a small amount of H_TwoWash with O Followed by washing with EtOAc and acetone to give a white solid product (9.2 mg) (Yield: 77%).¹HNMR (MeOH-d4,300MHz)   Result: Thrombin inhibitor al by binding polypeptide and chemically reactive group One-step derivatization of gatroban has been achieved. Simply put, the argatroban molecule Of the N-hydroxybenzotriazole ester Activation by HBTU by formation of a tell, which in turn binds to the amino group of the binding polypeptide To give the desired adduct. Argatroban molecular weight-equivalent to water molecule Byproducts with abundance were also observed by mass spectroscopy. This by-product is probably From internal nucleophilic attack on activated esters. Argatroban molecule is nucleophilic More by-products if pre-activated with HBTU before the addition of the reactive amino ester Would have been generated. Therefore, amino esters and argatroban are Mixed prior to HBTU addition. The product was purified on a preparative TLC plate. Yield ratio Relatively low (28-53%), but a simple one that does not protect these functional groups of argatroban Step binding polypeptide substitutions are very satisfactory.   Two amino fatty acids having a 6 methylene unit span and a 12 methylene unit span Methyl esters as binding polypeptides for their structural stability and simplicity I chose. Variable length binding polypeptides are involved in optimal drug presentation to its target Will provide information on length requirements.   Subsequent hydrolysis of the methyl ester of the binding polypeptide under alkaline conditions is circular. Proceeding smoothly to give the corresponding acid in 73-85% yield. N-hydroxysuccinyl Treatment of the free acid with HBTU in the presence of amide and DIEA reduces the desired NHS ester to 42% Given in yield. Then, using EDC as a coupling reagent, yield To 69%. NHS ester formation¹Confirmed by HNMR and mass spectroscopy .Example 2 :Derivatized argatroban molecule biology after covalent binding to rabbit erythrocytes Have a proper use .   Materials and Methods: Rabbit erythrocytes were converted to argatroban-C6 NHS ester compound (compound YL-015-35). To do this, rabbit erythrocytes are treated with fresh citrate. From isolated blood. Briefly, an aliquot of blood over 18 minutes Centrifuged at 00 rpm (Sorvall RC-5B, SH-3000 rotor). Buffy coat Pipette off the upper layer of the cell pellet and remove the remaining erythrocytes four times with PBS, pH 7.4. Washed. Remove aliquots of cells for counting before final wash did. 1 x 10 washed red blood cells⁹Cells / mL again. Sa The total volume of labeled cells per sample was 3 mL. Allow cells to reach room temperature for 45 minutes Reacted with one of the following reagents with gentle stirring across: (1) sham, 0 0.5% DMSO / PBS; (2) 100 μM argatroban C6-free acid in 0.5% DMSO / PBS ( Compound YL-015-15); (3) 10 μM argatroban C6-NHS in 0.5% DMSO / PBS Stele (Compound YL-015-35); (4) Argatroban C6 at 50 μM in 0.5% DMSO / PBS -NHS ester (compound YL-015-35); (5) 100 μM argato in 0.5% DMSO / PBS Roban C6-NHS ester (compound YL-015-35); and (6) 100 μM in 0.5% DMSO / PBS Of sulfo-NHS-LC-biotin (Pierce, Cat # 21335). After the labeling reaction, the cells Was centrifuged at 1800 rpm for 4 minutes and the supernatant was discarded. 0.5% DMSO / PBS 10m Resuspended in 1 l and pelletized. The cells were washed once more with 10 ml of PBS.   The cells were subsequently treated with 6 mg / mL of BS to quench any remaining reactive esters. Resuspend in A / PBS (Sigma, A-2934, Lot 93HO291) and allow 30 minutes at room temperature Gently. After stopping the reaction, the cells were washed five times with PBS. Fine cell yield It was measured by cell counting.   After binding, cells are hypotonically lysed and the resulting membrane (ghost) and cytosol Fractions were analyzed by immunoblotting and ELISA. For details, The combined red blood cell pellet was chilled on ice for 5 minutes. Then put the cells on ice Ice-cold 5 mM phosphate buffer containing 1 mM fefablock and 40 μM leupepsin It was dissolved in 12 ml of pH 8.0 (PB). Stir the lysate briefly, then at 4 ° C at 15,000 rpm Centrifuged for 20 minutes (SS-34, Soval RC-5B). Pellet supernatant (lysate) Were pipetted off, labeled and stored at 4 ° C. Pellet (ghost) with cold PBS12m Washed 4 times with l and centrifuged as above. Micro BCA Kit (Pierce; Cataro 23231) to reduce the protein content of ghosts and hemolysates. It was measured. Cell fractions were stored at -80 ° C.   The sample was then added to a 2X reducing cocktail (5% SDS in 0.25 M Tris-HCl, pH 6.8, 5 (0% glycerol, 0.5 M 2-mercaptoethanol). 100 ℃ Sample for 5 minutes and then briefly cooled, the sample was analyzed by SDS-PAGE for 10% Degraded with acrylamide mini gel. Electrophoretically transferred to nitrocellulose, After a short Ponceau S staining, the blots were blotted at room temperature for 2 hours ( 5% non-fat dry milk in PBS, pH 7.4). Then blot With TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.25% Tween 20). Wash twice and with a 1: 1000 dilution of rabbit serum in TBS-T at room temperature for 2 hours. Incubated. The blot was then washed four times with TBS-T as described above, With biotinylated anti-rabbit IgG antibody (Vector, BA-1000) at room temperature for Incubated. The blot was washed as above, then for 30 minutes. At room temperature with ABC-HRP (vector). After washing, blot Visualized with the Metal Enhancement DAB Substrate Kit (Pierce, Cat # 34065).   Results: The results of the above experiments show that a number of erythrocyte membrane proteins are derivatized -C6 NHS ester molecule. In contrast, The presence of the derivatized argatroban molecule of sol protein is at the limit of detection and this Thrombin inhibitor compounds bind covalently to proteins located outside the cell And ultimately suggested that it was an effective reagent for drug delivery. Furthermore, The capture ELISA assay shows that the portion of the thrombin inhibitor-labeled protein is It was observed that it belongs to the glycophorin family of proteins.Example 3 :Derivatives to human serum albumin (HSA) and rabbit serum albumin (RSA) Spectroscopic analysis of argatroban fluoride binding   Materials and Methods: Protein Human Serum Albumin (HSA) Using Mass Spectroscopy Binding of derivatized argatroban molecules to rabbit and rabbit serum albumin (RSA) Number of complex argatroban molecules not only supporting but also covalently attached to the protein Was quantified. In-line coupled to an electrospray ionization (ESI) mass spectrometer (MS) Using high performance liquid chromatography (HPLC), which is called HPLC / ESIMS , All samples were analyzed. In addition, matrix-assisted laser desorption ionization (M A-LDI) using time-of-flight (TOF) mass spectrometer (this is called (MALDI-TOFMS)) Then, several samples were analyzed.   HPLC / ESIMS samples are prepared as duplicate reactions or as described below. A portion of the same reaction sample used for the described ELISA assay. these Inject the sample directly into HPLC / ESIMS only, except for diluting with water. In the case of, PBS, DMSO, and unreacted derivative molecules are induced by protein-argatroban Separated from the conductor molecule complex. Samples for MALDI-MS were first purified by HPLC and 10 mg / ml cinnamin in 50% acetonitrile / 50% water on a stainless steel analytical plate Directly mixed 1: 1 with the matrix of pinic acid. Then the mixture is gentle with air Drying was performed with a gentle flow to enable crystallization and inserted into the apparatus for laser ionization.   HPLC Hewlett-Packard 1090 series with diode array detector Figure 2 is a liquid chromatogram of Size II. The column used for the separation was Brownley Aqua Pore OD-300 reverse phase C18 column, 1 mm × 10 cm, pore size 300 mm, particle size 7 μm. The mass spectrometer to which the HPLC is connected using the fused silica capillary tube is Ion atomization source to enable atmospheric pressure ionization of liquid flowing continuously into the device Is a Sciex API-300 triple quadrupole mass spectrometer equipped with MALDI-TOFMS is Perceptive Biosystems Voyager Elite driven in linear fashion It is a DE device.   result:   (1)Argatroban-C6 NHS ester vs Argatroban-C12 NHS ester, 20: 1 Molar excess of ester vs HSA or RSA   Argatroban-C6 NHS ester (Compound YL-015-35) at 100 μM for 1 hour at room temperature The HPLC / ESIMS of the reacted HSA (0.33 μg / μl, 5 μM) was 1-8 argatos per HSA molecule. The covalent addition of a loban derivative molecule was shown. 100 μM Argatroban-C6 NHS Esthe RSA (0.30 μg / μl, 4.5 μM) reacted with the compound (compound YL-015-35) for 1 hour at room temperature. The covalent addition of 1-8 argatroban derivative molecules per RSA molecule was shown. But Argatroban-C12 NHS ester (compound YL-015-11) under the same conditions Soon the 1-5 argatroban derivative is covalently added per HSA or RSA molecule. These results indicate that argatroban has a 6-carbon atom binding polypeptide. Argatroban derivatives in which the conductor molecule has a 12-carbon-atom binding polypeptide More effective on available reactive functionalities of both HSA and RSA than offspring It suggests that it acts to give a good bond.   (2)HSA vs RSA , Argatroban-C12 NHS ester, 1:10 mole shortage esthetic Le vs protein   0.5 μM argatroban-C12 NHS ester (compound YL-015-11) for 1 hour at room temperature The HPLC / ESIMS of the reacted HSA (0.33% μg / μl, 5 μM) was 0-4 alga per HSA molecule. The covalent addition of a troban derivative molecule was shown. 0.5 μM argatroban-C12 NHS RSA (0.30 μg / μl, 4.5 μM) reacted with stele (compound YL-015-11) for 1 hour at room temperature Is the covalent addition of 0-3 argatroban derivative molecules per RSA molecule, or HSA protein It showed slightly less covalent labeling than was obtained with quality.   (3)15 μM vs 30 μM RSA, argatroban-C6 NHS ester, 10-12: 1 molar excess Extra ester vs RSA   Argatroban-C6 NHS ester (compound YL-015-35) at 200 μM for 1 hour at room temperature HPLC / ESIMS of reacted RSA (1.0 μg / μl, 15 μM) was 1-9 argatos per RSA molecule. The covalent addition of a loban derivative molecule was shown. 300 μM Argatroban-C6 NHS Esthe RSA (2.0 μg / μl, 30 μM) reacted with the compound (compound YL-015-35) for 1 hour at room temperature The covalent addition of 1-9 argatroban derivative molecules per RSA molecule was shown. These results Was confirmed by MALDI-MS analysis of these two samples, which Shows a single broad peak concentrated on molecular weight. Based on these average molecular weights, 15 μM / 200 μM RSA / argatroban derivative samples were loaded with additional An average of 5.6 argatroban derivative molecules were generated (range 1-10 added) and 30μ The M / 300 μM RSA / argatroban derivative sample was the average added per RSA molecule. An argatroban derivative molecule of 5.4 was generated (range 1-10 added).   (Four)4.5 Concentration of 1-100 μM argatroban-C6 NHS ester reacted with μM RSA Degree study   Argatroban-C6 NHS ester (Compound YL-015-35) (1, 10, 15, 20, 25, and HPLC / ESIMS of RSA (0.30 μg / μl, 4.5 μM) reacted with room temperature for 1 hour at room temperature Is argatroban-induced for each molecule of RSA as shown by the results below A linear covalent addition of body molecules resulted.   1 μM does not add detectable argatroban derivative molecules.   10 μM adds one argatroban derivative molecule to about 50% of the RSA molecules.   15 μM adds 0-3 argatroban derivative molecules per RSA molecule (for RSA molecules) Most are +1).   20 μM adds 1-3 argatroban derivative molecules per RSA molecule (for RSA molecules) Most are +1 and +2).   25 μM adds 1-4 argatroban derivative molecules per RSA molecule (for RSA molecules Most are +1 and +2).   100 μM adds 2-10 argatroban derivative molecules per RSA molecule (the major species) None).Example 4 :To assess the relative bioavailability of derivatized HSA to thrombin Sandwich ELISA assay   Substances and methods: Human serum albumin (HSA, Sigma catalog at a concentration of 333 μg / ml) No. # A-8763) or rabbit serum albumin (RSAN Sigma Kata) at a concentration of 300 μg / ml. Log # A-9438) at room temperature for 1 hour in PBS, pH 7.4, 100 μM argato Roban-C6 or -C12 NHS ester (Compound YL-015-35 or YL-015-11, respectively) And reacted. Add 50 mM final concentration of hydroxylamine, pH 7.8, to the reaction And stopped by incubation for 10 minutes at room temperature. Pre-reaction stopped negative pair Control samples were treated with argatroban NHS ester for 10 min before addition of HSA or RSA. Except that the reaction was carried out with hydroxylamine for the same time. Most Later, HSA or RSA only samples were added with the addition of argatroban NHS ester solution. Add DMSO to 1% to simulate and incubate as for other reactions. And the reaction was stopped.   Rabbit anti-HSA (Boehringer / Mannheim, Cat. No. 605001) antibody or Dietary anti-RSA (Capel, Catalog No. 55629) antibody was diluted 1: 5000 in PBS, pH 7.4, Aliquot 0 μl / well into NUNC maxi soap F96 plate (Cat. No. 439454) I made a coat. Plates are incubated overnight at 4 ° C, then 1% BSA / PBS2 Blocked by addition of 00 μl / well and incubated for 1 hour at room temperature. Next The plates were then washed five times by immersion in PBS, pH 7.4.   Various HSA / argatroban derivative samples or RSA / argatroban derivative Samples were serially diluted in 1% BSA / PBS (1: 100 to 1: 50,000) and 100 μl / well Each sample dilution was added to duplicate wells. Samples on plate for 2 hours Incubate at room temperature for a second and then wash the plate as described above.   Thrombin (11.4 mg / ml through Enzymes Systems) with 0.1% BSA / 9.4 μg / ml in 0.1% PEG8000 (Sigma, Catalog No. P-2139) in PBS, pH 7. And 100 μl / well were added. Plates are incubated at room temperature for 1 hour. Cuvated and washed as above.   Mouse anti-toxin, an antibody that may bind away from the active site of thrombin Robin (American Diagnostics, Cat.No.EST-7) with 0.1% BSA / Dilute to 1 μg / ml in PBS, pH 7.4, containing 0.1% PEG 8000 and add 100 μl / well Was added to the wells. Incubate the plate at room temperature for 30 minutes, Washed as described.   Biotinylated rabbit anti-mouse IgG (mouse gamma specificity, Zymd, Cat. -6540) was diluted 1: 1000 in PBS, pH 7.4, containing 0.1% BSA and 1% PEG 8000, 100 μl / well was added to all wells. Plate at room temperature for 30 minutes Incubate and wash as above.   Next, 100 μl / well of ABC-HRP (vector, catalog number PK4000) was added, Dilute each component 1: 500 in PBS, pH 7.4 containing 0.1% Tween 20, before use For 30 minutes at room temperature. Add PEG 8000 directly to ABC-HRP Previously added to 0.1%. Plates are incubated with ABC-HRP at room temperature for 30 minutes. Incubate and then wash 10 times by immersion in PBS, pH 7.4.   Apart from adding biotinylated rabbit anti-mouse IgG and ABC-HRP, assay Simplified and diluted 1: 500 in HRP diluent (Medix, catalog number RIH4203) Goat anti-mouse IgG conjugated to HRP (Jackson Immunoresearch, Cataro Background number was reduced by adding μl / well was added. Plates are incubated for 30 minutes at room temperature, then PBS Washed 10 times by immersion in pH 7.4.   Finally, 100 μl / well of o-phenylenediamine dihydrochloride (OPD, Sigma, Catalog number P-3804) to 0.015% H_TwoO_TwoCitrate-phosphate buffer, pH 5.3 At 0.5 mg / ml and incubated for 30 minutes at room temperature. incubation Later, 200 μl / well of 2N sulfuric acid (VWR, catalog number VW73S00-1) was added and After stirring the plate for 5 seconds, the absorbance was measured using a SpectraMax plate reader (model). OD in Recursive Devices₄₉₀Read in.   Results: The results obtained from the above sandwich ELISA assay are shown in FIGS. Is shown. The data shown in FIG. 2 is for the argatroban NHS Argatroban-C6 or -C12NHS compared to samples containing Samples containing HSA reacted with esters (compounds YL-015-35 and YL-015-11, respectively) OD in wells incubated with₄₉₀Demonstrated a significant increase in I do. Significant signal on background is C6 derivatized compound and C12 derivative Was detected at approximately 10 ng / ml for both of the Nulls are very different, with C6 having a significantly higher OD (about 6-7 fold at saturation). these The data shows that HSA proteins can interact with and bind to thrombin. Argatroban NHS ester derivative molecule is covalently bound to HSA protein In turn, this suggests that this can be subsequently detected with anti-thrombin antibodies. Therefore These data show that argatroban derivatives covalently linked to the HSA protein It suggests that it can bind to and inhibit thrombin.   For RSA, only argatroban C6 NHS ester derivatives are in ELISA format Detected (see FIG. 3). Significant signal on background It was detected at about 30 ng / ml for the gatroban-C6 derivative. In this assay, absolute The target signal is lower than the signal obtained with HSA. This is the capture antibody, the degree of labeling And differences in availability, or both, due to differences in assays. May be.   The covalent bond between the argatroban NHS ester and the HSA or RSA protein is Mass spectrometry data (from the double reaction sample above or from the rest of the reaction sample) Argatroban derivative molecules in the sample Generated coadducts for most of the HSA or RSA proteins present That was shown.   Human glycophorin A protein (hGPA) is also used under the same conditions as the above RSA protein. Reacted with argatroban-C6 NHS ester derivative (compound YL-015-35) below. this The results of these studies are shown in FIG. As shown in FIG. 4, hGPA protein is Reacts with Lugatroban-C6 NHS derivative, binds thrombin by ELISA, approx. 30 ng / ml was shown to produce a detectable signal. ELISA assay is 0.1M vinegar Mouse anti-hGPA (Bio-Antrant) diluted to 10 μg / ml in sodium acid, pH 4.5 Block / CRTS, mAb 5F4) reacted with NHS-argatroban derivative Assays described above for HSA and RSA, except used to capture It was the same.   The results of the above experiments show that the argatroban derivative molecule of the present invention It not only binds covalently to reactive functional groups on protein but also binds to and inhibits thrombin. Clearly demonstrate doing so in a way that does not inhibit the ability of the derivative molecule .   Also, keyhole limpet hemocyanin (KLHN Sigma, catalog number R-5755) Is argatroban-C12 NHS ester (compound YL-015-11). Argatroban-C12 NHS ester (Compound YL-015 -11) was bound to thrombin by ELISA assay, It was shown to produce a detectable signal at 00 ng / ml. ELISA assay, PBS Rabbit anti-KLH (Capel, cat # 55966) diluted 1: 5000 in pH 7.4 -HS above, except used to capture KLH reacted with argatroban derivative Same as assay described for A and RSA. Extremely heterogeneous KLH formulation These properties make it impossible to analyze these samples by mass spectrometry. won. However, similar substances (2 mg / ml KLH and 3.3 μM argatroban -C12 NHS ester-containing reaction) used argatroban derivative polyclonal antibody To confirm the covalent modification of the KLH protein.Example 5 :HSA, hGPA and rabbit red blood conjugated with argatroban-C6 NHS ester Sandwich ELISA using polyclonal antibodies to evaluate ball ghosts Assay   Substances and methods: 300 μg / ml human serum albumin (HSAN Sigma, Cataro A-8763) or human glycophorin A (hGPA) protein for 1 hour in PBS, pH 7.4. Argatroban-C6 NHS ester (Compound YL-015-35) at room temperature over a ). Add hydroxylamine, pH 7.8 to a final concentration of 50 mM, and add The reaction was stopped by incubating at for 10 minutes. Pre-reaction stopped Negative control samples were argatroban-C6 NHS ester derivatives with HSA or hGPA After reacting with hydroxylamine for 10 minutes prior to protein addition, The outside was of the same composition. Finally, samples containing only HSA or hGPA Add DMSO to 1% to simulate the addition of the gatroban derivative solution, The reaction was incubated in the same manner as the reaction and stopped.   Rabbit anti-HSA monoclonal antibody (Boehlin diluted 1: 5000 in PBS, pH 7.4) Gar / Mannheim, cat. No. 605001) or mouse anti-hGPA monoclonal Body (BioAtlantis diluted to 10 μg / ml in 0.1 M sodium acetate, pH 4.5) / CRTS, mAb 5F4 or mAb 3C10 or 1: 100 diluted 1: 500 in PBS, pH 7.4 MAb 3E4 diluted to 0-each antibody recognizes a different epitope of hGPA) 100 μl / well using NUNC Maxi Soap F96 plate (Cat. No. 439454) Each protein was captured by making aliquots into each. Plate at 4 ° C Incubate overnight and then block by adding 200 μl / well of 1% BSA / PBS. Locked and incubated for 1 hour at room temperature. The plate is then washed in PBS, pH 7.4. Washed 5 times by immersion.   Various HSA / argatroban derivative samples or hGPA / argatroban derivative Samples were serially diluted (1: 100-1: 50,000) in 1% BSA / PBS and 100 μl / well Duplicate wells were added for each sample dilution. Sample on plate Incubate for 2 hours at room temperature, then wash as above.   Rabbit anti-KLH argatroban antibody at 1: 500 or 1% in 1% BSA / PBS, pH 7.4 Dilute 1: 1000 and add 100 μl / well to each well. Then remove the plate Incubate for 30 minutes at room temperature and wash as above.   Biotinylated goat anti-rabbit IgG (vector, catalog number BAI000) was added to 1% BSA / P Dilute 1: 1000 in BS, pH 7.4 and add 100 μl / well to all wells. Step The rate was incubated for 30 minutes at room temperature and washed as above.   Next, 100 μl / well of ABC-HRP (Vector, Catalog No.PK4000) was added, Dilute each component 1: 500 in 0.1% Tween 20 in PBS, pH 7.4, before use Incubated for 30 minutes at room temperature. The plate is then immersed in PBS, pH 7.4 Washed 10 times.   Finally, o-phenylenediamine dihydrochloride (OPD, Sigma, catalog number P-3804) ) 100 μl / well with 0.015% H_TwoO_TwoCitrate-phosphate buffer, pH 5.3 At 0.5 mg / ml and incubated at room temperature for 15-20 minutes. Then 100μl / Add 2N sulfuric acid (VWR, catalog number VW3500-1) to the wells and stir the plate for 5 seconds After reading the absorbance, use a Spectramax plate reader (Molecular Devices) in OD₄₉₀Read in.   Results: The results of the above experiments are shown in FIGS. 5, 6, 7 and 8. For details, With argatroban-C6 NHS ester or protein alone In comparison, sump reacted with argatroban-C6 NHS ester (compound YL-015-35) OD in wells incubated with₄₉₀Was significantly increased. Bag Significant signal on background is about 13 ng / m for HSA-argatroban derivative 1 (see FIG. 5) and for the hGPA-argatroban derivative ng / ml (5F4 mAb, see FIG. 6). Also, other anti-hGPA monoc Lonal antibodies (3C10 and 3E4) detected hGPA-argatroban derivatives, but at low levels. (See FIGS. 7 and 8, respectively). These data are Van-C6 NHS ester covalently binds to HSA and hGPA proteins When combined, a thrombin / antithrombin result indicating binding to thrombin Confirm.   Prepared with 100 μM argatroban-C6 NHS ester (Compound YL-015-35) Reacted Usaki erythrocyte ghosts are also polyclonal anti-KLH-Argatrova Plate is positive in ELISA format using detection antibody Rabbit erythrocytes reacted with 50 μM argatroban-C6 NHS ester Was tested, but the signal was very low). Sham-treated rabbit red blood Rabbit erythrocytes treated with spheres or argatroban-C6 free acid (compound YL-015-15) Produced no signal above background. Goat diluted 1: 500 Anti-rabbit erythrocytes (Capel, cat # 55616) or diluted 1: 100 in PBS, pH 7.4 Mouse anti-hGPA (bioatlantic, mAb 3C10) was argatroban-C6 NHS EL, except used to capture rabbit erythrocyte ghosts that reacted with esters The ISA assay is similar to the assays described above for HSA and RSA proteins. It was similar. Then, the polysaccharide is prepared in the same manner as the HSA protein and RSA protein Using clonal anti-KLH-argatroban antibody (1: 1000), argatroban-C6 NHS ester was detected. Argatroban-C6NHS ester (top HGPA labeled with the same substance as described above) in both assays and up to 60 ng / ml goat Detected by anti-rabbit erythrocytes and mouse anti-hGPA up to 30 ng / ml. Goat anti rabbit The rabbit erythrocyte / NHS-argatroban complex is captured using erythrocytes and It was detected as 1.6-3.2 μg / ml of protein. Using mouse anti-hGPA (mAb3C10) Capturing rabbit erythrocyte / NHS-argatroban complex into approximately 160 μg of total protein / ml.   These results indicate that argatroban-C6 NHS ester derivative was polyclonal anti-K Covalently bound to rabbit erythrocytes in a manner that is detectable using the LH-argatroban antibody Indicates that they have been combined. Since mouse anti-hGPA was successful in rabbit cells, this assay Argatroban-C6 NHS S covalently bound to human erythrocyte ghosts and all cells It should be able to detect tell. In addition, goat anti-rabbit erythrocyte antibody Perhaps this reagent was able to detect human erythrocyte ghosts and cells. Can be used as well.Example 6 :Detection of thrombin inhibitory activity   Argatroban derivatives that inhibit thrombin activity using various assays The ability was detected. Specifically, a Km-based chromogen assay was first used . First, thrombin induced addition in argatroban or a derivative thereof, 0.1% PEG. 8 μM S-2238 (H-D-Phe-Pip-Arg-p-nitroanili as a substrate for water splitting 2HCl), PBS, pH 7.2, 1% DMSO at 35 ° C in a total volume of 1 mL from human plasma Starting with the addition of 10 mU of thrombin and measuring the absorbance at 405 nm for 10 minutes. Specified. Measurements at 405 nm are for thrombin of p-nitroaniline from S-2238 substrate Designed to detect induced release. As such, at 405 nm The observed increase in absorbance is directly correlated with an increase in substrate hydrolysis by thrombin. There is a clerk. The results from these studies are based on the IC for underivatized argatroban.₅₀ Is 20 nM, while the IC for C12-linked carboxylate of argatroban₅₀ Was 150 nM.   Similar results indicate that 25 μM N-t-Boc-β-benzyl-Asp-Pro-Arg- 96-well format based on Km using 7-amido-4-methylcoumarin HCl (A total volume of 250 μl, 0.1 mg / ml BSA in 1% DMSO, 25 μM substrate in PBS was started by addition of 2.5 mU thrombin from human plasma and Luminous intensity was measured at 460 nm for 10 minutes). From these studies, the underivatized IC about Lugatroban₅₀Is 6 nM and the C12-linked carboxy of argatroban In terms of the rate, it is about 80 nM, and the compound C-conjugate of YL-015-15, argatroban The combined carboxylate was about 50 nM.   Vmax based assay (250 μl total volume, 0.1% hydrolyzed substrate in PEG 1% DMSO in 200 μM S-2238, PBS, pH 7.2, 25 ° C. as 40 m from human plasma Initiated with U thrombin and measured at 405 mn for 20 minutes) IC about Argatroban₅₀Was 250 nM. Argato in this assay IC for Loban's C12-linked carboxylate₅₀Was about 5-8 uM. C12-bond Argatroban derivatized with C6 instead of the free acid or methyl ester form 1.25-10uM IC, depending on₅₀Had.   Reaction conditions for binding of HSA by NHS ester derivative of argatroban: 5μ M HSA in PBS, pH 7.4, 1.5 DMSO, 1.5% ethanol at room temperature for 1.5 hours Was incubated with 235 μM of the NHS ester of argatroban. At this point, unreacted esters in the sample are added with 1.2 mM hydroxylamine. And stopped at room temperature for 30 minutes. The combined HSA is quicksep (kwiksep) The unbound form of the argatroban derivative is removed by desalting on a column and Centricon-1 Concentrated to near initial reaction volume using 0 column. 1) Underivatized HSA protein And 2) using HSA protein treated with argatroban NHS ester Approximately the same procedure was followed for the control.   In a Km-based suppression assay for thrombin from human plasma, PBS 2.5 mU thrombi in 10 μl of 0.1% polyethylene glycol 8000 (PEG) in pH 7.2 To a final concentration of 10 μM S-2238 hydrolysis substrate, 1% DMSO, 0.1% PEG, and And 40 μg / ml HSA or 240 μl PBS without HSA. Test HSA protein bound to argatroban NHS ester, argatroban HSA treated with NHS ester pre-quenched with bang hydroxylamine and As in the sample, the residual or free acid form of the NHS ester of argatroban Treated by the same incubation and desalting used to remove Contains HSA. The rate of substrate hydrolysis by thrombin is limited to free p-nitroani Initial in all cases, measured by the increase in absorbance at 405 nm from the formation of phosphorus It was almost linear for 10 minutes. 1) lacking HSA or 2) underivatized H HSA containing SA or 3) treated with argatroban quenched NHS ester In double wells containing, the rate of substrate hydrolysis was indistinguishable between samples and 0.94 It ranged from mOD / min to 1.08 mOD / min. Argatroban NHS ester In double wells containing combined HSA, the rate of substrate hydrolysis was 0.66 mOD / min to 0.71 m The range was up to OD / min and was thus reduced by 32% compared to the control.   In a second independent experiment, HSA was re-substituted with argatroban-C12 NHS ester (compound YL). -015-11). Also contains pre-reacted NHS ester or HSA alone A control was prepared. The samples were analyzed in duplicate and the concentration range was 20, 40, and 80 mg / ml. The enclosure was tested. HSA conjugated with argatroban-C12 NHS ester is a control sample 80-mg / ml inhibited thrombin activity by 35-42% compared to the control sample. The suppression was 15-20% with mg / ml.Example 7 :A Km-based fluorogen probe for thrombin suppression from human plasma Sussey   Thrombin from human plasma was transferred to Enzyme System Products (Dublin, Glycerol from California) in water 1: 1, 11.4 mg / ml, 3800 U / mg, MW 36,700 And stored undiluted at -20 ° C. It was freshly diluted on the day of use.   To dilute for the assay, add 0.67 μl thrombin (25 units) to cold PBS Buco, catalog number 14190-136, not containing calcium chloride and magnesium chloride 0.1 mg / ml BSA (Pierce, Cat.No. 23209, BSA fraction in pH 7.2) V, 2 mg / ml stock solution in saline containing sodium azide) After stirring for 5 hours, a stock solution of 5 mU thrombin / μl was obtained. 250 μl of this stock solution is 0.1 mg BSA 0.125mU / μl or 3.125mU / 25μ 1 was obtained.   The substrate is N-t-Boc-β-benzyl-Asp-Pro-Arg-7-amido-4-methylcoumarin HCl. Catalog number B-4028, lot 122H0070, F.W. Obtained as 770.3 . 5 mg of a 4 mM stock solution was dissolved in 1.42 ml of DMSO. Put the stock solution in the dark at -20 ° C Stored in 200 μl aliquots.   Another substrate is N-tBoc-Val-Pro-Arg-7-amido-4-methylcoumarin HCl, From catalog number B-9385, lot 53H0805, F.W. Obtained as 627.7 Was. 5 mg of a 4 mM stock solution was dissolved in 1.863 ml of DMSO. -20 ° C in the dark And stored in 200 μl aliquots.   The fluorescent standard was 7-amido-4-methylcoumarin, which was Log No. 25,737-0, MW 175.19. 6.1 mg of 20 mM stock solution in DMSO 1.7 Dissolved in 43 ml. The stock solution was stored in a 200 μl aliquot in the dark at −20 ° C.   The assay was performed in a 96-well well with Perceptive Bisystems Cytoflu-I. Crotiter plate (Nunc imino module maxisorb, flat bottom police) (Tylene plate) at ambient temperature (about 20 ° C.). Each well or Prepare a stock solution containing common concentrations and components for groups of wells, and add 200 μl Aliquots were made per microtiter well. Run the assay in a total volume of 250 μl. This is done routinely with 25 μl for inhibitor and thrombin. To allow 25 μl (about 3 mU).   For the initial determination of Km and Vmax, the fluorogenic substrate N-tBoc-β-benzyl-Asp-Pro The concentration of -Arg-7-amido-4-methylcoumarin was varied from 5 μM to 200 μM.   For the inhibition assay, the final concentration per well (250 μl V_T) Is 25 μl N-t Boc-β-benzyl-Asp-Pro-Arg-7-amido-4-methylcoumarin HCl (approximate Km), 0 It contained .1 mg BSA / ml PBS and 1% DMSO. Abnormal reading the initial reading Ensured the elimination of bad wells with high background (at 360 nM (λ excitation, 40 nM bandwidth, λ emission, 460 nm, 40 nm bandwidth). Then putative inhibitors Add 25 μl of 10X stock solution to the wells, mix for 5 seconds and let sit at ambient temperature for 10 minutes Dynamic reading and background interference and / or The increase in quality hydrolysis was evaluated.   Argatroban or a soluble binding variant of argatroban (C6 or C12) For the inhibition assay used, samples were dissolved in DMSO at 1 mM and then buffered as described above. Diluted in 1% DMSO in medium to suit the assay.   Bound to ghosts derived from red blood cells by RSA or activator Derivatized argatroban molecules were formulated in phosphate buffer. (1) Activate Argatroban molecules lacking a promoter or (2) Additional ghost samples treated with activator groups attached to gand RSA was included as a negative control.   Then add wells with 25 μl thrombin / multichannel pipettor The thrombin assay was started by adding and mixed for 5 seconds. Fluorescent products ( Free 7-amido-4-methylcoumarin released from peptide by thrombin ) Was tracked simultaneously in 48 wells (one reading per minute over 30 minutes, 360 readings). excitation in nm, bandwidth 40 nm, emission 460 nm, bandwidth 40 mn). Reaction speed Is measured as the initial velocity for the first 10 minutes, and the initial background Relative fluorescence units (RFU) of about 9,000-10,000 unsuppressed reactions over und reading Signal (80 gain setting). Routinely, 5 of the initial substrate % Are converted to fluorescent products in 10 minutes, and the samples and curves are run in duplicate. It is. Standard curve of thrombin activity from 0.3125-3.5 mU / well, as well as 7-amide- Standard curve for 4-methylcoumarin (1% DMSO, 0.1 mg BSA / ml, PBS, V_T250μl / Range from 5 to 200 pmol / well). The pH of each sample is At the end of the assay, pH was found to be 7.2 on paper.   The results of the above assay are shown in FIGS. Specifically, FIG. 9 shows thrombin 4 shows the ability of RSA-argatroban (C6) complex to suppress. IC for this complex₅₀ Was estimated to be 25 nM. In contrast, the control RSA sample was The degree was not inhibitory. The result is a C6 bond covalently linked to the RSA protein Argatroban molecules are not only bioactive, but also bioavailable. Confirm. In fact, this observation suggests that erythroid cells labeled with C6-linked argatroban molecules (See Figure 10). This graph shows 100 μM Argatro Increased ghosts derived from erythrocytes labeled with van (C6) -NHS ester Figure 3 shows a recoat kinetic assay. Control sham-treated ghost or equivalent Ghosts derived from cells treated with gatroban C6-carboxylate are suppressed. Not controlled (data not shown). Together, these days Argatroban molecule with cell-related or non-cell-related carrier protein Is an effective drug carrier when bound to Demonstrate maintaining the biological activity of the bitter.Example 8 :Platelet aggregation assay   Chosen Argatroban (C6) -RSA Conjugate Chosen Thrombin-induced Platelet Aggregation Assay The body's biological activity was confirmed. The conjugate was prepared according to the following protocol. In PBS Argatroban-C6 NHS at room temperature for 1 hour at room temperature Reacted with ester. Sham samples were incubated at RT for the same time in 2% DMSO in PBS. Consists of RSA incubated over length. Then Amicon Centri Using a Con 30 filtration / concentrator, each of the samples was concentrated to a final volume of 1.5 mL. Was. The concentrated sample is then pierced with 5 mL of Quick Sep Ex Cellulose Plus It was desalted on a tick desalting column. The waste containing RSA or argatropane-C6-RSA is then The removed volume was subjected to BCA microprotein estimation and lyophilization. In addition, this The samples were analyzed by LC / MS, sandwich ELISA assay (described above) Thrombin binding and immunoblotting were measured. From LC / MS analysis, average 10 Argatroban molecules were bound to each RSA molecule.   Platelets were diluted with anticoagulant buffer ACD (9 mL of blood per mL of buffer) Purified from freshly drawn human blood. Blood at 200xg for 25 minutes Centrifuged. Platelet-rich plasma (PRP) was collected with a plastic pipette. PRP was centrifuged at 3,000 xg for 15 minutes in a v-shaped plastic tube. Next Remove the supernatant (referred to as platelet-poor plasma (PPP)) with, save and buffer platelets Solution A1 (35 mM citric acid, 103 mM NaCl, 5 mM glucose and KCl, 2 mM Ca Cl_Two, 1 mM MgCl_Two3.5 mg / mL BSA, 0.1 μM PGEI and 0.3 U / ml apyrase, p H6.5). The resuspended platelets were washed twice with buffer A1. After the last wash, platelets were washed with buffer B1 (137 mM NaCl, 2.6 mM KCl, 12 mM NaHCO3)._Three , 0.3 mM NaH_TwoPO_Four3 mM CaCl_Two1 ml of MgCl_Two5 mM glucose, 3.5 mg / mL 3 x 10 in BSA and 0.05 U / ml apyrase, pH 7.4)^Fiveresuspend at a density of / μl Was. Platelets were kept at room temperature until ready for use.   Various parameters related to platelet aggregation were analyzed using AFFI B10 Obtained with a tunnel aggregometer. Briefly, warm the reaction chamber to 37 ° C Stir at a speed of 1200 rpm. Pass light through the reaction chamber and add 300 μl of buffer B1. Use to set 100% transmission level for each chamber. Series of markers From the leveling experiment, human thrombin (Sigma) 0.5 U / ml is the optimal aggregation concentration of thrombin And used to set the instrument to 0% transmission. It is. The standard conditions for the assay are that 250 μl of the platelet suspension is With 50 μl of inhibitor diluted in buffer at 37 ° C. for 0 seconds, and Involves incubating without an inhibitor. The transmittance value is now Set as 0%. After this measurement, thrombin is added to the reaction chamber. (20 μl), measure the transmittance over the next 6 minutes. Experiment with 3 replicate samples did.   FIG. 11 shows the delay of thrombin-induced platelet aggregation as affected by various titrations of RSA. Show. Argatroban-C6-RSA, in contrast to sham-treated RSA, has platelet aggregation Obviously delayed.   Table 1 shows the estimated concentration of the complex that inhibits 50% of aggregation (IC₅₀) Is displayed.                                 Table 1 Compound IC ₅₀ Free argatroban 0.1-0.15μM RSA-Argatroban-C6 complex 4.5-7.5 μM RSA sham> 150μM   As shown in Table 1, an average of 10 argatroban / RSA and RSA Putative IC for Argatroban-C6-RSA using known dilutions₅₀Measured the value However, it was in the range of 4.5 to 7.5 μM. In contrast, sham-treated RSA was 150μ Even M did not interfere with platelet aggregation. Higher concentrations were not tested and IC for Jam RSA₅₀Was not measured. Argatroban in this assay IC for₅₀Is about 0.1 μM, which is in agreement with literature values. This value is Argat It is at least as effective as Roban-C6-RSA complex. This tendency is based on the Km -Based fluorogenic thrombin inhibition assay compared to argatroban in C6- Enhanced IC observed for bound argatroban carboxylate₅₀Based on Was expected.   These results indicate that the present invention provides a greatly improved treatment with thrombin suppression. It is clear that With the use of the present invention, complex thrombin inhibitors For an extended period of time, so that repeated dosing is not required and No compliance by the operator is required and protection is ensured. The present invention Derivatized thrombin inhibitors of erythrocytes, plasma proteins and various other Covalently binds to vascular components of the plant, retains biological activity and is not immunogenic .   All publications and patent applications mentioned in this specification are as if each As specifically and individually indicated for inclusion in the article or patent application by reference To the extent included herein by reference.   Since the present invention has now been fully described, many changes and modifications depart from the scope of the claims. It will be apparent to one skilled in the art that the present invention may be practiced without.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/06 A61K 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, I , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Ezrin Allen M United States California 94556 Moraga Quintas Lane 110 (72) Inventor Song Yanghon 94404 Foster City, Barclay, The Drive 1125 Apartment K 309

Claims (1)

[Claims] 1. A compound of the following formula: Wherein Y is a linking group consisting of 2-30 atoms, and Z is a chemically reactive group capable of reacting with a reactive functional group of the target molecule in an aqueous system to form a covalent bond therewith, or It is a precursor that can be activated to a chemically reactive group, and the compound has thrombin inhibiting activity in vivo when bound to a long-lived blood component. 2． 2. The compound according to claim 1, wherein Y is an alkylene group, an oxyalkylene group or a polyoxyalkylene group. 3． The compound according to claim 1, wherein Y is an alkyl group. Four. 4. The compound according to claim 3, wherein said alkyl group has 6 carbon atoms. Five. 4. The compound according to claim 3, wherein said alkyl group has 12 carbon atoms. 6． The compound according to claim 1, wherein Z is a carboxy group, a carboxyester group or a mixed acid anhydride. 7． The compound according to claim 6, wherein Z is selected from the group consisting of N-hydroxysuccinimide, isocyanate, thiol ester, thionocarboxylic acid ester, imino ester, carbodiimide anhydride, carbonate ester and phosphoryl ester. 8． The compound according to claim 1, wherein Z is N-hydroxysuccinimide. 9． A composition comprising the compound according to claim 1 and a physiologically acceptable carrier. Ten. A method of inhibiting thrombin activity in vivo, said method comprising introducing the compound of claim 1 into the bloodstream of a mammalian host in an amount sufficient to provide an effective amount for thrombin inhibition. Wherein the compound reacts with a long-lived blood component and becomes covalently bound, and the thrombin inhibition is maintained for an extended period of time relative to the lifetime of the unbound thrombin inhibitor. . 11． 11. The method of claim 10, wherein said introducing is intravascular. 12． A method for inhibiting thrombin activity in vivo, said method comprising: (a) separating blood or blood components from a mammalian host; and (b) separating said blood or blood components with a compound as defined in claim 1 and ex. contacting in vivo (the compound covalently binds to one or more reactive functional groups present in the blood or blood component), and (c) the covalently bound blood or blood obtained in step (b) Introducing a component into the blood stream of said mammalian host, wherein the thrombin inhibition is maintained for an extended period of time compared to the lifetime of unbound thrombin inhibitor. 13. 13. The method according to claim 10 or claim 12, comprising the steps of: removing a blood sample from said mammalian host; and analyzing for the presence of said compound bound to a long-lived blood component. 14. A blood portion comprising the compound of claim 1 covalently linked to at least one long-lived blood component. 15． 15. The blood component according to claim 14, wherein the long-lived blood component is an immunoglobulin. 16． 15. The blood portion according to claim 14, wherein the long-lived blood component is a red blood cell. 17． A conjugate comprising a compound according to claim 1 covalently bound to a blood component selected from the group consisting of serum albumin, immunoglobulin and erythrocytes. 18． 18. The complex according to claim 17, wherein the blood component is serum albumin.